INTRODUCTION

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide,in which statistically 3.2% of men and 2.6% of women will die from the disease[1,2].CRC has a survival rate of 91% if detected in stage 1.However,its overall 5-year survival rate is only 65%,according to 2020 data from the American Cancer Society published on the SEER database[3].Surgical removal of rectal cancer remains the firstline treatment of CRC.However,non-surgical treatment options serve as important treatment tools,as rates of screening and surgery approvals between various nations can lead to differences in rate of survival[4].

Molecular hydrogen (H) has been studied extensively as a therapeutic gas,with an estimated 1500 publications to date exploring its potential therapeutic use in 170 disease models across every organ in the mammalian body.Hcan be administered through several methods,such as Hinhalation,dissolving Hgas in water to make hydrogen-rich water (HRW) for oral consumption or topical application,or hydrogenrich saline.

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5-fluorouracil (5-FU) is a widely used chemotherapeutic employed during cancer treatment[5].Hhas shown positive effects in terms of quality of life in human clinical research.For example,studies report that Htherapy was associated with improved liver function in patients who were administered chemotherapy,as well as reduced side effects for those receiving radiation therapy,and protective effects against radiation-induced bone marrow damage in cancer patients[6-8].

Hhas been previously demonstrated to display anti-cancer properties when administered on its own.Hyperbaric Htherapy has been examined as a potential cancer therapy,revealing potent anti-tumor effects in mice with squamous cell carcinoma[9]In a study involving mice with colon cancer,it was shown that drinking HRW dose-dependently potentiated the tumor-inhibitory activity of 5-FU by enhancing cellular apoptosis of the cancer cells[10].In the present study,we aimed to explore the potential effects of a higher concentration of HRW than previously utilized,to further explore the effects of high-concentration HRW compared to control,5-FU administration on its own,as well as HRW in combination with 5-FU,for the mitigation of CRC progression and accompanying outcomes.

MATERIALS AND METHODS

Chemicals and reagents

HRW was created using H-producing tablets (HRW Natural Health Products Inc.,New Westminster BC,Canada) by dissolving it in a 500-mL beaker.HRW was made two times each day every 12 h.The concentration of HRW was＞1.5 mmol/L and remained＞0.1 mmol/L after 12 h as determined by redox titration (H2Blue;H2Sciences,Las Vegas,Nevada).5-FU was obtained from EBEWE Pharma,Unterach,Austria.F12/Dulbecco’s Modified Eagle Medium (DMEM/F12),fetal bovine serum(FBS),penicillin (Pen) and streptomycin (Strep) were obtained from Gibco BRL,Life Technologies Inc.(Gaithersburg,MD,United States).

Cell culture

The mouse colorectal adenocarcinoma cell line CT-26 was obtained from Pasteur Institute (Tehran,Iran).CT-26 cells were cultured in DMEM/F12 medium containing 10% FBS,Pen (50 U/mL) and Strep (50 μg/mL) in a humidified atmosphere containing 5% COand 95% air at 37 °C.

I stood before him, stunned11 and silent. Finally, after all these years, my heart joined my head in understanding. My father loved me so much that just saying so made him weep, which was something he never, ever wanted to do, least of all in front of family. Mom had been right. Every day of my life Dad had told me how much he loved me by what he did and what he gave. I know, Dad, I said. I know. And now at last I did.

Xenografts in mice:Treatment and evaluation

Tumor xenograft experiments were conducted as previously described by Golovko[11].In brief,6-to 8-wk-old female inbred Balb/c mice were injected with 5 × 10CT-26 cells (100 μL) into the left rear flank (day 0).When tumor volumes reached 80-100 mm(-10 d),24 mice bearing tumors were divided randomly into four groups (=6 per group) and treated as follows:(1) The control group;(2) The 5-FU group received intraperitoneal injections of 5-FU (5 mg/kg) every other day;(3) The Hgroup received HRW both from drinking water and by delivering 200 μL of the solutionoral gavage;and (4) The combination group,H(administered in same way as group three) combined with 5-FU (administered in the same way as group two).The tumor volume was calculated every other day according to the following formula:V=(length × width)/2[12].The animals were sacrificed on day 14 and the tumors were removed for further analysis.

Histological assay

Fixed tumor tissue samples were embedded in paraffin wax and then sectioned at 5 μm thickness with a microtome.The tumor tissue sections were deparaffinized and stained with Hematoxylin-Eosin for evaluation of tumor necrosis.Masson trichrome staining was also performed for evaluation of collagen content and fibrosis.

The colon tissues samples were homogenized in ice with PBS and centrifuged.The supernatant was stored at-70 °C for the determination of the oxidative and antioxidative proteins.

When administered on its own,HRW demonstrated similar benefits in reducing tumor size compared to 5-FU.These results corroborate earlier reports that HRW can suppress early tumor formation in rats[24].Additionally,molecular Hwas shown to prevent tumor progression in a cell line model of lung cancer[25].In our study,we demonstrated that HRW administration was associated with a significant decrease in pathological collagen content equivalent to that of 5-FU.In contrast to previous reports demonstrating that molecular Hupregulates collagen biosynthesis and expression,and corresponding to the results of another study reporting that molecular Hsignificantly reduced type III collagen depositions as observedstaining[26-28].Molecular Hhas shown to both be able to promote and suppress outcomes,model dependent,for many biological processes,which may indicate that contradictory reports do not undermine our understanding of the mechanisms by which Hoperates.HRW demonstrated similar outcomes to 5-FU for visual results of fibrosis from mass trichrome staining.Further,molecular Hhas been previously demonstrated to reduce fibrosis in the lungs and abdomen[29,30].

Tissue preparation for measurement of oxidative stress markers

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Malondialdehyde measurement

So far,little is known regarding the effects of various dosages and different administration methods of HRW,Hinhalation,and hydrogen-rich saline on cancer cells.To date,several routes have shown potential benefits of HRW administration for cancer treatment,including the previously cited reports using inhalation studies in humans,and HRW use in murine models.Additionally,a recent report demonstrated that the use of H-producing reactive magnesium implants was associated with significant suppression of tumor growth in a mouse model of ovarian cancer[43].However,since Hhas demonstrated protective effects on healthy cells,it could also protect and stimulate cancer cell growth.For example,Hadministration has been demonstrated to induce the mitochondrial unfolded protein response,which is also a proliferative signal in various cancer cells[44,45].Therefore,the effects of different dosages and administration methods of molecular Hshould be carefully analyzed to determine its effects.

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Total thiol group measurement

The Mashhad University of Medical Sciences Committee on Animal Ethics has approved all animal protocols used in this research.Reference Number:991229;Date:July 10,2020.

Evaluation of superoxide dismutase and catalase

Superoxide dismutase (SOD) was determined with a colorimetric assay described by Madesh[15].The method is centered on the synthesis of SOD by pyrogallol autooxidation and inhibition of superoxide-dependent reduction of 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) to its formazan.Catalase was measured by evaluating the kinetics of HOhydrolysis at 240 nm in a buffer of sodium phosphate.The velocity of the enzyme activity can be determined by converting HOto HO and Owithin 60 s of normal conditions[15].

Ethics statement

We used di-thio nitrobenzoic acid (DTNB) reagent for measurement of total thiol group as previously described[13].Briefly,1 mL of Tris-EDTA buffer (pH=8.6) was added to the colon homogenate and absorbance was measured.Similarly,20 μL of DTNB reagents was added to the sample absorbance and the absorbance was measured again;subsequently,the total molar concentration of thiol was determined as previously described[14].

Data analysis

The statistical methods of this study were reviewed by Dr.Mohammad Taghi Shakeri,a member of the Biostatistic Department of Mashad University of Medical Sciences.All the results are presented as means and standard error of the mean (mean ± SEM).The differences in the mean values among different groups were determined by a one-way analysis of variance (ANOVA) using the SPSS 22.0 program.Significance was set at values of＜0.05.

RESULTS

H2 suppresses tumor growth and enhances the antitumor efficacy of 5-FU in a colon cancer xenograft model

We studied the influence of Hon tumor growth in a CRC xenograft model.Administration of HRW significantly decreased tumor growth in mice (Figure 1).The suppressive effect of HRW on tumor growth was slightly,but not statistically,more potent than 5-FU,and not as effective as the combination therapy.Specifically,the average control tumor size was 2698.85 mm,whereas the average tumor size in the HRW group was 2047.23 mm(24.1% suppression compared to control),while the average tumor size in the 5-FU group was 2097.32 mm(22.3% suppression compared to control).The combination group of HRW + 5-FU resulted in the greatest tumor size suppression,with the average size of 1177.5 mm(56.4% suppression compared to control) (Figure 1A).

Similarly,a comparison of tumor weight between the groups showed that both 5-FU and HRW significantly reduced tumor weight (＜0.05),and this decrease was potentiated in the combination group of HRW + 5-FU (＜0.001) (Figure 1B).Treatment with 5-FU reached statistical significance at day 12 through 14 (＜0.05) as compared to control,whereas the combination treatment of HRW + 5-FU reached significance by day 6 (＜0.05) and continued its suppressive effect at day 8 (＜0.01)and days 10-14 (＜0.001) compared to control.Moreover,combination treatment was more effective compared to 5-FU treatment alone,reaching significance by day 8 (＜0.05) with days 10 through 14 being even more significant (＜0.01).

The effects of H2 and 5-FU on redox status

We investigated the effects of Hadministereddrinking HRW on levels of markers of OS in tissue homogenates.As shown in Figure 2,HRW treatment decreased MDA levels in tumor tissues compared to control (＜0.05) and 5-FU treatment (＜0.001).However,5-FU treatment increased MDA levels compared to control (Figure 2A,＜0.001).HRW tended to improve activity of all three antioxidant markers measured.For example,HRW increased thiol concentrations (Figure 2B) compared to control (＜0.01) and 5-FU treatment (＜0.001).Additionally,we observed a trend towards an increase in SOD and catalase activity following Htreatment,although significance was only reached when compared to 5-FU treatment.Compared to the control group,there was a significant decrease in levels of all antioxidant markers following 5-FU treatment.In the combination group (HRW and 5-FU),a more prevalent rise in OS and suppression of AA was observed compared to 5-FU alone.MDA levels significantly increased in the combination group compared to control (Figures 2B,2C and 2D;＜0.001) and the 5-FU treatment (P＜0.05).Similarly,activity of all three antioxidants measured were suppressed by the combination compared to control and also when compared to 5-FU alone.

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H2 and 5-FU increased necrotic areas

Tumor necrosis was observed under a light microscope.As illustrated in Figures 3A and 3B,treatment of Hor 5-FU displayed interspersed tissue necrosis compared to the untreated group.In the H+ 5-FU group,we observed larger necrotic areas than the necrotic areas in either group alone.

H2 and 5-FU decreased tumor fibrosis in the colon cancer xenograft model

New treatments with potential positive effects in CRC are desperately needed,particularly treatments with high safety profiles and low side effects.HRW may fit the criteria as a safe potential treatment for CRC,either as a stand-alone treatment or in combination with conventional treatments.

DISCUSSION

Non-surgical treatment options to improve outcomes in CRC remains a high priority.Ideal adjuvant treatment options should aim to improve quality of life,reduce symptoms,and work synergistically with standard care.Although 5-FU remains the front-line treatment option for a variety of cancers due to its effectiveness,it also has limitations.For instance,cardiotoxicity has shown to be a serious side effect of 5-FU administration,largely due to increases in OS and suppression of endogenous antioxidant mobilization[16].Accordingly,molecular Hhas been proposed as a novel approach for the treatment of cardiovascular disorders due to its ability to significantly reduce the effects of OS[17].

Our results demonstrate that the combination of HRW and 5-FU treatment potentiated the beneficial anti-tumor effects of both treatments on their own,such as tumor weight,size,the degree of fibrosis and collagen content in the tumor.Enigmatically,while treatment with HRW on its own significantly improved all three measured antioxidant markers while decreasing levels of MDA,the combination therapy of HRW and 5-FU significantly blunted AA and elevated MDA levels significantly above those measured with 5-FU alone.Acute temporal increases in OS after Hadministration have been noted in other studies,and it has been previously suggested that molecular Hmay act as a therapeutic hormetic agent similar to exercise[18-21].Nogueira[19] (2018) examined the effects of molecular Hadministration on exercise performance and noted an acute rise in OS in the Htreated group,followed by a greater antioxidant mobilization,leading to improved redox homeostasis shortly after the exercise period ended.Further,previously published human clinical research has demonstrated significant improvements in redox homeostasis following medium-term administration of HRW for 24 wk[22].Since our short-term study was unable to determine the effects of H+ 5-FU administration on OS and AA over a longer treatment course,such as has been reported in previous research on HRW[23],future research is warranted to investigate this area.

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Interestingly,the combination of 5-FU and HRW demonstrated significant reductions of tumor weight,size,collagen deposition and degree of fibrosis,while increasing markers of OS and blunting AA significantly beyond 5-FU alone.Previous studies have demonstrated that HRW generally reduces OS in most animal and human disease models when administered as a stand-alone intervention.Molecular Hhas been observed to work in an additive or synergistic capacity with several other interventions in various models,as demonstrated by a recent study,in which administration of high-concentration HRW alongside minocycline improved outcomes following ischemic stroke in rats[31].Additionally,molecular Hhas shown to enhance the effects of photothermal therapy by inhibiting tumor progression in cell cultures and was also shown to act equivalently to sulfasalazine in a dextran sodium sulfate-induced mouse model of colitis,with the combination therapy of HRW +sulfasalazine demonstrating effects of significantly greater magnitude than either treatment on its own[32,33].

Treatment with 5-FU has been shown to result in DNA damage[34].Alterations in various processes,such as nucleotide and amino acid metabolism,may lead to 5-FU resistance[35].Autophagy plays an important role in nucleotide and amino acid metabolism,and Hhas been shown to both stimulate and mitigate autophagy for beneficial outcomes[36-40].It has also been suggested that therapies which mediate DNA repair alongside 5-FU and other cancer treatments should be further explored as a therapeutic target[41].For instance,it has been shown that Hexerted significant protective effects against DNA damage in calf thymus tissue following exposure to radiation[42].Future research is also needed in order to address both potential protective effects and long-term benefits of molecular Hdelivered in conjunction with 5-FU and other conventional cancer therapies,exploring outcomes related to DNA damage,cell signaling,and survival rates.

Malondialdehyde (MDA) was measured by methods as previously described[13].Briefly,1 mL of homogenate was mixed with 2 mL of a solution containing thiobarbituric acid,trichloroacetic acid,and HCl in hot water (100 °C) for 45 min and centrifuged for ten minutes.The MDA levels were determined by measuring the absorbance of the solution.

CONCLUSION

Safe and well-tolerated adjuvant therapeutics with the potential to ameliorate the deleterious consequences of various cancer treatments,while simultaneously improving outcomes,are of high interest to cancer patients and the medical community.Molecular Htherapy demonstrates potential anti-cancer properties,as well as the ability to reduce the secondary effects of various treatments.In this study,we have shown that administered on its own,HRW demonstrates anti-cancer properties and improves markers of OS and AA compared to conventional treatment(5-FU).The combination of HRW and 5-FU suppressed tumor progression in a synergistic manner;however,the addition of HRW to 5-FU treatment increased OS levels and reduced AA.Limitations of this study include that the observation period during the study was only 14 d,with rates of survival and remission not examined.As such,interpretation of these results should be evaluated cautiously.Larger,longerterm studies are highly warranted to explore HRW as an adjuvant therapy for various cancers,alongside conventional therapy,with longer observational periods needed to address unanswered questions regarding potential positive and negative effects of molecular Hon redox homeostasis during cancer treatment.

ARTICLE HIGHLIGHTS

Research background

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide.Surgical removal remains the first-line treatment for CRC;however,nonsurgical options remain important tools for treatment.Currently,treatments such as 5-fluorouracil (5-FU),a widely administered chemotherapeutic agent utilized in the treatment of CRC,presents known beneficial effects,but also significant side effects.Hydrogen-rich water (HRW) has demonstrated beneficial effects in numerous species,including humans,in many disease models,including various cancers.One attractive aspect of HRW is the high safety profile and low rates of side effects combined with its promising therapeutic effects.

Research motivation

We used Masson’s trichrome staining to compare the collagen deposition in tumor tissues across the treatment and control groups.Our results demonstrate that administration of either Hor 5-FU suppressed collagen deposition and degree of fibrosis compared to the control group (Figure 4A).The increment in percentage of collagen deposition in all treated groups was significantly decreased when compared to the control group (Figure 4B;＜0.001).Specifically,collagen deposition percentage in the control group was 24.6%.In contrast,with both Hand 5-FU alone,the percentage of collagen deposition in the tumor tissue was significantly reduced to about 13% (＜0.001).However,administration of both H+ 5-FU in combination further reduced the percentage of collagen deposition in tumor tissue (=3%) compared to both the 5-FU group and Hgroup (＜0.001).

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Research objectives

We aimed to evaluate the efficacy of HRW on a CRC model compared to 5-FU and control,as well as the combination treatment of HRW and 5-FU compared to 5-FU alone,HRW alone,or control.We measured tumor size,tumor weight,fibrosis,and collagen content,as well as oxidative stress (OS) and antioxidant activity (AA) in mice with induced CRC.These objectives allow us to determine the therapeutic efficacy and mechanistic insight of HRW with or without 5-FU,as well as determine if there are additive benefits in a combinational treatment to guide future clinical studies.

Research methods

Six-to eight-week-old female inbred Balb/c mice were injected with 5 × 10CT-26 cells(100 μL) into the left rear flank (day 0).When tumor volumes reached 80-100 mm,24 mice bearing tumors were randomly divided into four groups.Mice were either left untreated (control) or treated with 5-FU (intraperitoneal injection,5 mg/kg every other day),high-concentration HRW produced by magnesium tablets (ad libitum in drinking water,as well as by oral gavage 200 μL daily),or both HRW and 5-FU.

Research results

We report that molecular hydrogen dissolved in water (HRW) was as effective as 5-FU,with more preferential outcomes relating to higher AA and lower OS.Importantly,the combination of HRW and 5-FU was superior to either therapy on its own,presenting the possibility that HRW may be explored as an adjuvant therapy alongside conventional chemotherapeutics.

Research conclusions

HRW may be a novel safe adjuvant therapy for treating CRC,either as a stand-alone therapy,or preferably,alongside conventional chemotherapeutics.

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Research perspectives

Clinical research to evaluate the effects of HRW as a treatment for CRC,both alone and in combination with 5-FU and other chemotherapeutics,is highly warranted.

We thank Mr.Alex Tarnava,CEO of HRW Natural Health Products Inc.for kindly donating Drink HRW tablets for this study and Mashhad University of Medical Sciences for their support.